Tsüklilise adenosiinmonofosfaadi biosensori arendamine ja rakendamine G valguga seotud retseptorite
signaaliülekande uurimiseks
Maailma rahvastik on pidevas kasvus kuid ka vananemises ning sellest on tingitud mõnd haigust või sündroomi põdevate patsientide üha suurenev hulk.
Enamik haigustest on põhjustatud häiretest organismi metabolismi või rakkude signaaliülekande regulatsioonis. G valguga seotud retseptorid (GPCR) moo-dustavad suure rakkudevaheliste signaalide ülekannet reguleeriva valkude pere-konna. Inimese genoomist on leitud ligi 800 erineva GPCR-i geenijärjestused.
Keemilise signaaliülekande moduleerimine on levinud ravistrateegia, mida kin-nitab ka tõsiasi, et ligi kolmandik kõigist tänapäeva retseptiravimitest on suu-natud just GPCR-tele. Ravimid ehk bioaktiivsed ühendid on seega muutunud tänapäeva maailma asendamatuks osaks.
Uute molekulide otsinguil on väga olulisteks ühendite molekulaarne disain, farmakokineetiliste parameetrite ja bioloogilise aktiivsuse määramine ning katsetulemuste võrdlus struktuurselt sarnaste või kehaomaste signaalimoleku-lide omadustega. Ühendite süstemaatiliseks testimiseks on loodud mitmeid katsesüsteeme ning nende arendamine on väga aktuaalne ka täna. Käesolevas doktoritöös arendati välja katsesüsteem GPCR-te ligandide iseloomustamiseks lähtudes tsüklilise adenosiinmonofosfaadi (cAMP) signaaliraja aktivatsiooni jälgimisest.
cAMP on signaalimolekul, mis kannab rakus edasi paljude erinevate GPCR-de poolt vahendatud bioloogilisi teateid. Kui retseptorile seondunud ligand on aktiveeriva iseloomuga, suureneb või väheneb rakus cAMP sünteesi eest vastu-tavate ensüümide adenülaadi tsüklaaside aktiivsus ning muutus cAMP kont-sentratsioonis on võrdeline retseptori aktivatsiooniga. Rakendades cAMP suhtes tundlikke ja spetsiifilisi sensoreid, on võimalik jälgida retseptorile seostuvate ligandide bioloogilist aktiivsust elusrakkudes reaalajas. Käesolevas töös kasu-tatud biosensorid koosnevad cAMP siduvast peptiidijärjestusest ning kahest erinevate spektraalsete omadustega fluorestsentsvalgust. Puhkeolekus toimub kahe fluorofoori vahel Försteri resonantsenergia ülekanne (FRET). Ergastades biosensori fluorofoore kindla lainepikkusega valgusega (430 nm) on resonants-energia ülekande tulemusena võimalik määrata mõlema fluorofoori poolt kiira-tava valguse intensiivsust lainepikkustel 480 ja 530 nm. cAMP taseme suurene-misel rakus seostub signaalimolekul sensorile ning muudab fluorofooride oma-vahelist kaugust ja orientatsiooni võrreldes puhkeolekuga. Selle tulemusena väheneb fluorestsentsvalkude vaheline FRET ning muutub vastavatel laine-pikkustel detekteeritava valguse intensiivsuste suhe. FRET-l põhineva bio-sensoriga on seega võimalik mõõta cAMP muutust elusrakkudes vastusena raku pinnal asuvate retseptorite aktivatsioonile.
Biosensorite kasutamiseks on vajalik nende ekspressioon (rakud peaksid tootma sensorvalku ise, lähtudes seda kodeerivast geenijärjestusest).
Saavuta-maks biosensori ühtlast ekspressiooni erinevates uuritavates rakkudes loodi käesolevas töös bakuloviirustel põhinev BacMam ekspressioonisüsteem. Inime-sele ohutud putukaviirused transpordivad sensorit kodeeriva geenijärjestuse uuritavasse rakku, kus sellest sünteesitakse cAMP biosensorvalk. BacMam süsteem võimaldas reguleerida biosensori ekspressioonitaset, mille tulemuseks oli ühtlane ja hea korratavusega ekspressioon, mis lõi eelduse usaldusväärse katsesüsteemi loomiseks.
Töö käigus ekspresseeriti cAMP biosensorit mitmetes erinevates rakuliinides ning uuriti kolme erineva GPCR-i aktivatsiooni (melanokortiinne retseptor MC1R, dopamiini retseptor D1R ning inimese koorioni gonadotropiini ja luteni-seeriva hormooni LH retseptor). Need retseptorid seovad struktuurselt väga erinevaid ligande. MC1 retseptori ligandideks on peptiidid, D1 retseptor seob madalmolekulaarseid ühendeid ja LH retseptori ligandideks on mitmekümne kilodaltoni suurused glükoproteiinidest hormoonid. Leiti, et FRET sensoril põhinev katsesüsteem on sobilik kõigi nende GPCR-de ligandide bioloogilise aktiivsuse iseloomustamiseks, kusjuures süsteem võimaldab eristada nii osalisi kui ka täisagoniste (aktivaatoreid). Kuna loodud katsesüsteem on rakendatav nii cAMP tõusu kui languse määramiseks, siis võimaldas see määrata ka erinevate dopamiini retseptorite alatüüpe, mis nii aktiveerivad kui ka inhibeerivad ensüümi adenülaadi tsüklaas. Lisaks ligandide endi aktivatsioonile võimaldas katsesüsteem määrata ka ligandi seostumist moduleerivate komponentide mõju.
Näidati, et 1 mM Ca2+ või Mg2+ on vajalikud, et peptiidsed agonistid oleksid MC1 retseptoril efektiivsed. Katsesüsteemi tundlikkus (avastamispiir 5 pM) võimaldab seda kasutada aktiivsete hormoonide hulga määramiseks erinevates bioloogilistes proovides.
Käesolevas töös arendati välja FRET biosensoril põhinev katsesüsteem G valguga seotud retseptorite poolt reguleeritava cAMP taseme määramiseks elusrakkudes. Töö tulemusena on loodud tööriist, mille abil on võimalik mää-rata erinevate struktuursete omadustega ühendite bioloogilist aktiivsust vasta-vate retseptorite aktivatsiooni kaudu.
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